Mitochondrial Oxidative Phosphorylation: Interaction of Lead and Inorganic Phosphate 1

Abstract

Several of the physiological and biochemical defects observed in the liver, kidney, and erythropoietic tissue of lead poisoned animals have been associated with altered mitochondrial structure and function (1-3). These observations suggested a direct effect of lead on the functional integrity of mitochondria. Reticulocytes incubated with 100 μM lead acetate (PbAc) for 3 hr were found to have a significant inhibition in O₂ uptake. Exposure of actively respiring reticulocyte mitochondria to the same concentration of lead caused an immediate drop in oxidative activity (4, 5). Our preliminary studies with beef heart mitochondria indicated that the presence of inorganic phosphate in the incubation medium interfered with the interaction of lead with the mitochondrial membrane (6, 7). These findings, and the scarcity of information on the specific effects of lead on mitochondrial respiration, prompted an investigation of the dose-response relationship between lead concentration and mitochondrial oxidative phosphorylation in the presence and absence of exogenous inorganic phosphate.

Materials and Methods. Beef heart mitochondria were prepared by Nagarse digestion in a solution containing 250 mM sucrose with 1.0 mM ethyleneglycol-bis(beta-aminoethyl ether)-N,N'tetraacetic acid (EGTA), and buffered at pH 7.0 with tris(hydroxymethyl)aminomethane (Tris) (8). The mitochondrial samples were washed twice with Tris-sucrose solution to avert chelation of EGTA with the lead ion. The incubation medium utilized for the respiratory measurements contained 250 mM sucrose, 10 ml K₂HPO₄ (Pi), and 10 mM sodium succinate as substrate, adjusted to pH 7.4 with Tris-chloride (Tris-Cl). Oxygen uptake was recorded with a Yellow Springs Instrument Company Model 53 oxygen electrode system in 4 ml of air-equilibrated medium (9, 10). Respiratory measurements were made at 25°, immediately on addition of 2.5 mg of mitochondrial protein to the reaction chamber. The initial slopes of the oxygen electrode tracing were used to determine control values for succinate oxidation (State 4), adenosine diphosphate-stimulated respiration (State 3), and adenosine diphosphate to oxygen (ADP:O) ratios.